Materials Science and Engineering Program, University of California, Riverside.
Materials Science and Engineering Program, University of California, Riverside; Department of Bioengineering, University of California, Riverside; Stem Cell Center, University of California, Riverside;
J Vis Exp. 2022 Apr 15(182). doi: 10.3791/63065.
Over the past several decades, biodegradable materials have been extensively explored for biomedical applications such as orthopedic, dental, and craniomaxillofacial implants. To screen biodegradable materials for biomedical applications, it is necessary to evaluate these materials in terms of in vitro cell responses, cytocompatibility, and cytotoxicity. International Organization for Standardization (ISO) standards have been widely utilized in the evaluation of biomaterials. However, most ISO standards were originally established to assess the cytotoxicity of nondegradable materials, thus providing limited value for screening biodegradable materials. This article introduces and discusses three different culture methods, namely, direct culture method, direct exposure culture method, and exposure culture method for evaluating the in vitro cytocompatibility of biodegradable implant materials, including biodegradable polymers, ceramics, metals, and their composites, with different cell types. Research has shown that culture methods influence cell responses to biodegradable materials because their dynamic degradation induces spatiotemporal differences at the interface and in the local environment. Specifically, the direct culture method reveals the responses of cells seeded directly on the implants; the direct exposure culture method elucidates the responses of established host cells coming in contact with the implants; and the exposure culture method evaluates the established host cells that are not in direct contact with the implants but are influenced by the changes in the local environment due to implant degradation. This article provides examples of these three culture methods for studying the in vitro cytocompatibility of biodegradable implant materials and their interactions with bone marrow-derived mesenchymal stem cells (BMSCs). It also describes how to harvest, passage, culture, seed, fix, stain, characterize the cells, and analyze postculture media and materials. The in vitro methods described in this article mimic different scenarios of the in vivo environment, broadening the applicability and relevance of in vitro cytocompatibility testing of different biomaterials for various biomedical applications.
在过去几十年中，可生物降解材料在骨科、牙科和颅颌面植入物等生物医学应用中得到了广泛的探索。为了筛选生物医学应用的可生物降解材料，有必要根据体外细胞反应、细胞相容性和细胞毒性来评估这些材料。国际标准化组织（ISO）标准已广泛应用于生物材料的评估。然而，大多数 ISO 标准最初是为评估不可降解材料的细胞毒性而建立的，因此对于筛选可生物降解材料的价值有限。本文介绍并讨论了三种不同的培养方法，即直接培养法、直接暴露培养法和暴露培养法，用于评估不同细胞类型的可生物降解植入材料的体外细胞相容性，包括可生物降解聚合物、陶瓷、金属及其复合材料。研究表明，培养方法会影响细胞对可生物降解材料的反应，因为它们的动态降解会在界面和局部环境中引起时空差异。具体来说，直接培养法揭示了直接接种在植入物上的细胞的反应；直接暴露培养法阐明了与植入物接触的已建立的宿主细胞的反应；暴露培养法评估了与植入物没有直接接触但受植入物降解引起的局部环境变化影响的已建立的宿主细胞。本文提供了这三种培养方法的实例，用于研究可生物降解植入材料的体外细胞相容性及其与骨髓间充质干细胞（BMSCs）的相互作用。还描述了如何收获、传代、培养、接种、固定、染色、表征细胞以及分析培养后介质和材料。本文描述的体外方法模拟了体内环境的不同情况，拓宽了不同生物材料的体外细胞相容性测试在各种生物医学应用中的适用性和相关性。
